The present invention relates to a method for identifying an endocrine disruptor using a cell line having aromatase activity, particularly, a human granulosa-like tumor cell line; and a kit for carrying out the method.
The use of tributyltin (TBT) as a biocide in antifouling paints and wood preservatives leads to the contamination of the marine and freshwater environment with TBT. TBT has been shown to be highly toxic to a number of aquatic animals. Especially in marine prosobranch snails, TBT induces reproductive abnormalities and sterilization in female animals. This phenomenon, which has been called either pseudohermaphroditism or imposex, is characterized by the development of male sex organs (penis and/or vas deferens and prostate tissue) on females (Bryan et al, J. Mar. Biol. Ass. UK, 66:611-640 (1986); and Gibbs et al, J. Mar. Biol. Ass. UK, 66:767-777 (1986)). Not only TBT, but also triphenyltin (TPT) has been shown to have a strong effect on the development of imposex in the rock shell, Thais clavigera (Horiguchi et al, Environ. Pollution, 95:85-91 (1997)). However, the detailed biochemical mechanism of this phenomenon remains obscure. The involvement of sex steroids in the expression of imposex in marine neogastropods has been suggested based on the fact that pure female displays the lowest testosterone content, whereas advanced imposex stages have the highest testosterone content (Bettin et al, Helgolander Meeresunters, 50:299-317 (1996)), as well as the fact that the TBT-induced imposex is completely suppressed by antiandrogen cyproterone acetate, which is a competitive inhibitor of androgen receptors (Bettin et al, supra). TBT-induced imposex is mediated by an increasing androgen levels relative to estrogen levels, thus suggesting a decreased conversion of androgens to estrogens, i.e., aromatization (Bettin et al, supra; and Spooner et al, Mar. Environ. Res., 32:37-49 (1991)). This has been further supported by the fact that a specific aromatase inhibitor, SH489, exhibited the same imposex-inducing effect in neogatropods as did TBT-exposure (Bettin et al, supra). On the other hand, there is a controversial report which demonstrated no decrease in the activity of aromatase in gastropods, which were contaminated and exhibited clear evidence of imposex (Morcillo et al, Environ. Res., 81:349-354 (1999)). There is yet no clear direct in vitro evidence demonstrating that TBT compounds truly affect aromatase activity in any species, including marine species, as well as in humans.
A low dose of Dioxin or PCB, is known to cause problems in sperm formation in males that affects rates of hypospadias and cryptorchism. An orchioncus rise results which is passed to the next generation. Further, it is known that the pesticide Benomyl, can cause man sterility.
Thus, long-term and excessive exposure to endocrine disruptors in humans causes clinical problems, which relate to excessive androgens and/or reduced estrogens. Clinical problems in women include ovarian dysfunction, osteoporosis and hirsutism. Clinical problems in men include sterility. Hence, it is important to be able to identify endocrine disruptors so that exposure to the same can be eliminated.
A steroidogenic human ovarian granulosa-like tumor cell line, KGN, from a patient with invasive granulosa cell carcinoma has been established (Nishi et al, Endocrinol., 142:437-445 (2001); which is incorporated by reference herein in its entirety). The cell line possesses properties very similar to normal ovarian granulosa cells, including the expression of functional FSH receptor and a relatively high aromatase activity.
The KGN cell line was found in the present invention to be a useful model for investigating the in vitro effects of various compounds on aromatase activity in the mammalian system, and thus useful in identifying compounds which are effective as endocrine disruptors.
An object of the present invention is to provide a cell line useful for investigating the effects of compounds on aromatic activity.
Another object of the present invention is to provide a method for identifying an endocrine disruptor.
Still another object of the present invention is to provide a kit for carrying out said method.
These and other objects of the present invention, which will be apparent from the detailed description of the present invention provided hereinafter, have been met in one embodiment by a method for identifying an endocrine disruptor comprising the steps of:
(a) incubating a cell line having aromatase activity with a test compound;
(b) assaying for inhibition or activation of aromatase activity due to said test compound so as to identify an endocrine disruptor.